One of the limitations on the use of blood in an emergency setting is a requirement to type and cross-match the blood to minimize the risk of transfusion reactions. Type and cross-matching may require at least 10 minutes and a complete type and cross-match can take up to an hour. Furthermore, the risk of HIV transmission has been estimated to be 1 in 500,000 units of blood and the risk of hepatitis C transmission has been estimated to be 1 in 3,000 units. The safety of blood supply and blood logistics are critical issues in developing countries, where the risk of infectious disease transmission as well as the risk of outdated supply is relatively higher. Up to 25% of the blood is discarded in developing countries because of the presence of infectious disease. Hence, there are pressing factors to find blood substitutes or artificial blood compositions that avoid disease transmission and provide rapid response to improve chances of survival.
Two aspects of artificial blood use in clinical settings are volume expansion and oxygen therapeutics. Volume expander agents are inert, merely increasing, blood volume, and thus allow the heart to pump fluid efficiently. Oxygen therapeutics mimic human blood's oxygen transport ability. Oxygen therapeutics can be divided in two categories based on transport mechanism: perfluorocarbon based, which function by simple dissolution of oxygen, and hemoglobin protein based, which transports oxygen by facilitated capture and release. In hemoglobin-based products, pure hemoglobin (Hb) separated from red blood cells (RBCs) may not be useful for a number of reasons, including instability, induction of renal toxicity, and unsuitable oxygen transport and delivery characteristics when separated from red blood cells.
Hemoglobin based oxygen therapeutics have been shown to exert various degrees of vasoactive effects both in animal and human studies (Winslow et al., Adv Drug Del Rev 2000; 40: 131-42; Stowell et al., Transfusion 2001; 41: 287-99; Spahn et al., News Physiol Sci 2001; 16: 38-41; Spahn et al., Anesth Analg 1994; 78: 1000-21; Kasper et al., Anesth Analg 1996; 83: 921-7; Kasper et al., Anesth Analg 1998; 87: 284-91; Levy et al., J Thorac Cardiovasc Surg 2002; 124: 35-42). This vasoactivity may be due to the effects of these products in binding intracellular NO (Kasper et al., Anesth Analg 1996; 83: 921-7; Dietz et al., Anesth Analg 1997; 85: 265-273; Schechter et al., N Engl J Med 2003; 348: 1483-5), endothelial release (Gulati et al., Crit. Care Med 1996; 24: 13747), or sensitization of peripheral α-adrenergic receptors (Gulati et al., J Lab Clin Med 1994; 124: 125-33). Alternatively, the increased vasoconstrictive effects could be due to increases in the rate of oxygen release, secondary to the administration of these products, at a higher concentration than RBCs, resulting in vasoconstriction (Winslow et al., J Intern Med 2003; 253: 508-17; McCarthy et al., Biophys Chem 2001; 92: 103-17; Intaglietta et al., Cardiovasc Res 1996; 32: 632-43; Vandegriff et al., Transfusion 2003; 43: 509-16).
The ability of stroma-free Hb solutions to induce blood pressure increases has been known. It has been demonstrated that some cross-linked Hb solutions could increase mean arterial pressure as much as 25-30% in a dose-dependent manner within 15 min of administration and that the effect could last as long as 5 h.
Vasoconstriction may be due to NO scavenging by the hemoglobin based therapeutic (Katsuyama et al., Artif Cells Blood Substit Immobil Biotechnol 1994; 22: 1-7; Schultz et al., J Lab Clin Med 1993; 122:301-308, hereby incorporated by reference in its entirety). Vasoconstriction could be also caused by the contamination of the hemoglobin by phospholipids and endotoxin. Although the remaining phospholipids and endotoxin contamination during Hb purification may cause hemodynamic effects (Macdonald et al., Biomater Artif Cells Artif Organs 1990; 18: 263-282), it is less likely that this contamination be the major factor explaining the potent vasoactive effect of some of these products (Gulati et al., Life Sci 1995; 56: 1433-1442).
NO is a smooth-muscle relaxant that functions via activation of guanylate cyclase and the production of cGMP or by direct activation of calcium-dependent potassium channels. The increase in the free Hb can result in an increase in the NO binding. The increase in the NO binding can result in transient and in repeat dosing, sustained hemodynamic changes responding to vasoactive substances or the lack of vasoactive regulatory substances. In some circumstances the lack of nitric oxide may lead to blood pressure increases and if prolonged, hypertension. It has been demonstrated that NO may bind to the reactive sulfhydryls of Hb and may be transported to and from the tissues in a manner analogous to the transport of oxygen by heme groups (Jia et al., Nature 1996; 80:221-226).
Nitric oxide along with precapillary sphincter movement are regulators of the arteriolar perfusion of any tissue. Nitric oxide is synthesized and released by the endothelium in the arterial wall, where it can be bound by hemoglobin in red blood cells. When a tissue is receiving high levels of oxygen, nitric oxide is not released and the arterial wall muscle contracts making the vessel diameter smaller, thus decreasing perfusion rate and cause a change in cardiac output. When demand for oxygen increases, the endothelium releases nitric oxide, which causes vasodilatation. The nitric oxide control of arterial perfusion operates over the distance that NO diffuses after release from the endothelium. Nitric oxide is also needed to mediate certain inflammatory responses. For example, nitric oxide produced by the endothelium inhibits platelet aggregation. Consequently, as nitric oxide is bound by cell-free hemoglobin, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A2 and serotonin. These compounds may act synergistically with the reduced nitric oxide levels caused by hemoglobin scavenging resulting in significant vasoconstriction. In addition to inhibiting platelet aggregation, nitric oxide also inhibits neutrophil attachment to cell walls, which in turn may lead to cell wall damage. Because nitric oxide binds to hemoglobin inside the red blood cell, it is expected that nitric oxide may bind to free Hb (stroma free crosslinked tetrameric Hb) as well.
In many formulations free Hb and stabilized hemoglobin infusions appear to be linked to vasoconstriction of the blood vessels, resulting in extremely high blood pressures. The hemoglobin moiety of these products can diffuse into the endothelial lining of the vascular wall and act as a sink in binding and removing NO which is needed for maintaining the normal tone of the vascular wall. This can result in vasoconstriction of the smooth muscle cells of the vascular wall. The free Hb solution can leak into the surrounding tissues. Also, the extent of vasoconstriction which occurs subsequent to administration of different molecular size hemoglobin-based therapeutic bears an inverse relationship to the molecular size of the product used, i.e. infusion of larger oxygen carriers results in less vasoconstriction and hypertension (Sakai, et al. Am J Physiol 2000; 279: H908-15). The smaller sized Hb molecule may be the most permeable and may show a higher level of vasoconstriction and hypertension (Faivre-Fiorina et al., Am J Physiol Heart Circ Physiol 1999; 276: H766-70). In rabbit models, transfusion of free Hb through the ear vein has caused cerebral vasculature ischemia and death. Therefore, it is important to minimize the impact of administration of most free Hb on the arterial system during administration. Vasoactive agents such as verapamil, atenocard, sildenafil citrate, etc., may be administered to the patient prior to free Hb infusion. This is intended to ensure that the arterial system is minimally changed during infusion. Nitric oxide and verapamil are preferred vasoactive agents. Slow channel calcium blockers (or a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5), such as sildenafil citrate) may also be helpful in the prevention of the severe vasoconstriction. However, a slower infusion rate may not be possible with respect to a trauma patient when demand for volume is acute and critical.
One mechanism of modifying the NO scavenging properties of hemoglobin based therapeutics is blocking of NO binding sites on these molecules. Unprotected thiol on the cysteine moiety of the hemoglobin may bind with NO. Protection of thiol or sulfhydryl groups in the hemoglobin molecule may prevent the binding of NO to the hemoglobin at the thiol site and hence prevent an acute vasoactive response of the blood vessels causing a hypertensive reaction. The prevention of NO binding to hemoglobin based therapeutics may also prevent interference with normal platelet aggregation and neutrophil migration when this class of therapeutics is administered.
Therefore, some of the desirable characteristics of hemoglobin based oxygen delivery therapeutics are: toxicity-free, lack of induction of harmful immunogenic response, satisfactory oxygen carrying and delivery capacity, suitable circulatory persistence to permit effective oxygenation of tissues, long shelf life, capacity for storage at room temperature, absence of viral or other pathogens to prevent disease transmission, elimination of the requirement for blood typing, and capacity for deployment by first responders such as, paramedics, front line military medics etc. These characteristics provide a rapid, safe response to blood loss and the immediate support of tissue metabolic needs, thus improving the chances for survival.
The present invention disclosed herein provides compositions, characteristics and methods to prepare deoxygenated, endotoxin free, stroma free, thiol blocked, cross-linked tetrameric hemoglobin which has low reactivity with Nitric Oxide (NO), and the tetrameric structures is stabilized by cross-linking. In particular a carboxamidomethylated cross linked tetrameric hemoglobin is provided as a stable NO blocked tetrameric Hb of the invention, as well as methods for its production. A process and methods of preparation of stable NO-blocked tetrameric Hb of the invention are disclosed as well as methods of use as blood volume expansion agents and as oxygen delivery therapy agents.